All intracellular pathogens possess specialized strategies for invading their host cell, evading its defenses, and multiplying at the expense of its metabolism. The long term goal of the research described in this proposal is to understand the molecular basis of these events in a specific host-parasite system. To do this an extensive molecular genetic analysis has been undertaken of a facultative bacterial pathogen. Legionella pneumophila, the causative agent of legionnaire's disease and related respiratory ailments. This organism invades human macrophages and monocytes, evades their microbicidal systems, and grows exponentially within a specialized vacuole. These events are also observed with other intracellular pathogens. This system is ideally suited to molecular genetic analysis because L. pneumophila can be grown on bacterial culture media but grows exclusively intracellularly in tissue culture conditions. The products of genes required for monocyte killing (vir genes) will be identified. In some cases the nucleotide sequence of the vir genes will be determined. The structural gene for the major extracellular protein, a metalloprotease of broad specificity, (mep) will be sequenced and the regulation of mep expression will be studied using lac gene fusions. The conditions inside the monocytes during L. pneumophila infection will be probed with lac gene fusions to L. pneumophila "indicator genes" that are regulated by a variety of factors including: amino acid availability, oxidative damage, metabolic stress, iron availability, and osmolarity. Finally genes that are induced or repressed during monocyte infection will be identified by comparing the activities of lac fusions during growth in bacteriologic media versus growth in monocytes.